WO2016048105A1 - Composition for preventing or treating peripheral vascular disease using hepatocyte growth factor and stromal cell derived factor 1α - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating peripheral vascular disease, the composition comprising, as an active ingredient: (a) hepatocyte growth factor (HGF) or an isoform thereof, and stromal cell derived factor 1α (SDF-1α); or (b) a polynucleotide encoding the HGF and a polynucleotide encoding the SDF-1α. The peripheral vascular disease (for example, ischemic limb disease) can be more effectively prevented or treated through the significant promotion of vascular endothelial cell migration and angiogenesis in the case of singly using the composition of the present invention than in the case of using HGF, an isoform thereof, SDF-1α or a polynucleotide codes a protein thereof.

Description

Composition for the prevention or treatment of peripheral artery disease using hepatocyte growth factor and stromal cell inducer 1 alpha

The present invention provides a hepatocyte growth factor (HGF) or a variant thereof, and stromal cell derived factor 1 alpha (SDF-1α); Or it relates to a composition for the prevention or treatment of peripheral arterial disease comprising a polynucleotide encoding the proteins as an active ingredient.

This patent application claims priority to Korean Patent Application No. 10-2014-0129361 filed with the Korean Patent Office on September 26, 2014, the disclosure of which is incorporated herein by reference. Cardiovascular disease (Cadiovascular disease) is a disease caused by narrowing or occlusion of blood vessels due to atherosclerosis, and can be largely divided into coronary artery disease (CAD) and peripheral artery disease (PAD). have. Among these, a representative type of peripheral artery disease is ischemic limb disease.

To date, the treatment of cardiovascular diseases is mainly made up of the use of drugs to expand blood vessels and surgical operations. However, in case of ischemic retardation, the rotting pain is so severe that some patients take anti-systemic painkillers and severely cut their legs or even die, requiring fundamental treatment.

On the other hand, expression vectors as gene carriers for gene therapy are described in WO 2000/040737. In addition, WO 2003/078568 describes the effect of treating ischemic lower limb disease using the HGF gene.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made diligent research efforts to develop drugs that can prevent or treat peripheral arterial disease. As a result, Hepatocyte Growth Factor (HGF) or a variant thereof, and Stromal Cell derived Factor 1 alpha (SDF-1α); Alternatively, when the polynucleotides encoding the proteins are used together, the present invention is completed by confirming that a therapeutic effect on peripheral arterial disease is more remarkable than when using HGF, SDF-1α or polynucleotides encoding the proteins alone. Was done.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of peripheral artery diseases.

Another object of the present invention to provide a method of preventing or treating peripheral artery disease (Peripheral Artery Disease).

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a method for preparing an antibody comprising (a) Hepatocyte Growth Factor (HGF) or a variant thereof, and Stromal Cell derived Factor 1α (SDF-1α); Or (b) provides a pharmaceutical composition for the prevention or treatment of peripheral arterial disease comprising a polynucleotide encoding HGF and a polynucleotide encoding SDF-1α as an active ingredient.

The present inventors have made diligent research efforts to develop drugs that can prevent or treat peripheral arterial disease. As a result, Hepatocyte Growth Factor (HGF) or a variant thereof, and Stromal Cell derived Factor 1 alpha (SDF-1α); In addition, when the polynucleotides encoding the proteins are used in combination with HGF, SDF-1α or polynucleotides encoding proteins thereof, it was confirmed that the therapeutic effect on peripheral arterial disease is more remarkable.

The therapeutic strategies of the present invention can be broadly classified into two groups: protein therapy and gene therapy. According to the protein therapeutic strategy of the present invention, HGF or a variant thereof, and SDF-1α protein are used in combination. Meanwhile, according to the gene therapy strategy of the present invention, one or more nucleotide sequences encoding HGF and one or more nucleotide sequences encoding SDF-1α are administered. One or more nucleotide sequences encoding HGF and one or more nucleotide sequences encoding SDF-1α may be provided as one polynucleotide or may be provided as separate polynucleotides. According to one embodiment of the invention, one or more nucleotide sequences encoding HGF of the invention and one or more nucleotide sequences encoding SDF-1α are provided as separate polynucleotides.

Hereinafter, the present invention will be described in detail.

The term “isoform of HGF” as used herein has an amino acid sequence that is at least 80% identical to a naturally occurring HGF amino acid sequence in an animal, including all allelic variants. HGF polypeptide. For example, HGF variants have the meaning encompassing both the normal form or wild type of HGF, and various variants of HGF (eg, splicing variants and deletion variants).

As used herein, the term "prevention" refers to any action that inhibits or delays the progression of peripheral arterial disease by administration of a composition of the present invention.

The term “treatment” as used herein refers to (a) inhibition of the development of peripheral arterial disease; (b) alleviation of peripheral arterial disease; Or (c) elimination of peripheral arterial disease.

According to one embodiment of the invention, the HGF of the invention comprises recombinant human HGF protein. According to another embodiment of the present invention, the HGF of the present invention comprises the amino acid sequence of SEQ ID NO: 1 sequence.

According to one embodiment of the present invention, the HGF isoforms of the present invention include full length HGF (flHGF) and deleted variant HGF (dHGF).

As used herein, the term “flHGF” refers to amino acid 1-728 sequence of animal HGF, in one embodiment amino acid 1-728 sequence of mammalian HGF, and in another embodiment to amino acid 1-728 sequence of human HGF. .

As used herein, the term “dHGF” refers to a deleted variant of an HGF protein produced by selective splicing of an animal HGF gene, according to one embodiment a mammalian HGF gene. According to another embodiment of the present invention, the dHGF of the present invention is a human consisting of 723 amino acids lacking 5 amino acids (F, L, P, S and S) in the first Kringle domain of the alpha chain from the full length HGF sequence. HGF.

According to an embodiment of the present invention, the full length HGF of the present invention comprises the amino acid sequence of SEQ ID NO: 2, and the deleted variant HGF of the present invention comprises the amino acid sequence of SEQ ID NO: 3.

According to one embodiment of the invention, SDF-1α of the present invention comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 8 sequence.

As demonstrated in the following examples, when the HGF and SDF-1α proteins of the present invention were combined with HUVEC, the degree of vascular endothelial cell migration and angiogenesis was more effectively promoted than when each protein was treated alone. The combination of HGF and SDF-1α protein was confirmed to be effectively used for the prevention or treatment of peripheral arterial disease.

According to one embodiment of the invention, the variants of HGF of the invention are encoded by separate nucleotide sequences or by a single polynucleotide. The pharmaceutical composition of the present invention comprises two or more polynucleotides when the variant of HGF is encoded by a separate polynucleotide, and the single polynucleotide when the variant of HGF is encoded by a single polynucleotide. At least one polynucleotide. The polynucleotides of the present invention may be operably linked to one or more regulatory sequences (eg, promoters or enhancers) that regulate the expression of HGF isoforms.

If variants of HGF are encoded by separate polynucleotides, expression cassettes can be constructed in two ways. According to the first method, an expression cassette is constructed by connecting an expression control sequence to a CDS (coding sequence) for each of the variants. According to the second method, an expression cassette was constructed using an internal ribosomal entry site (IRES) or 2A peptide in the same manner as "expression control sequence-first heterologous CDS-IRES-second heterologous CDS-transcription sequence". do. IRES allows the translation of a gene to begin in the IRES sequence so that two or more genes of interest are expressed in the same construct.

When an isoform of HGF is encoded by a single polynucleotide, the polynucleotide encoding all of these isoforms is operably linked to a single expression control sequence.

In the present invention, a variant of HGF may be encoded by a hybrid HGF gene which simultaneously expresses two or more different types of variants, such as flHGF and dHGF.

According to one embodiment of the present invention, the hybrid HGF gene corresponds to a sequence corresponding to exons 1 to 4 of the human HGF gene, an intron 4 or a fragment sequence thereof, and an exon 5 to 18 of the human HGF gene. Sequence.

The hybrid HGF gene including the intron 4 is 7113 bp in length and includes the nucleotide sequence of SEQ ID NO: 7. The hybrid HGF gene may optionally comprise a fragment of intron 4 between exons 4 and 5 of HGF cDNA. According to a specific embodiment of the present invention, the hybrid HGF gene comprises the nucleotide sequence of SEQ ID NO: 5.

The “HGF isoform” of the present invention, a hybrid HGF gene (eg HGF-X7), has been reported in WO 2003/078568, the disclosure of which is incorporated herein by reference in its entirety.

The amino acid or nucleotide sequence of the HGF isoforms usable in the present invention is to be interpreted to include amino acid or nucleotide sequences that show substantial identity with the sequence of wild type human HGF isoforms. The substantial identity is minimal when the amino acid or nucleotide sequence of the wild-type human HGF isoform is aligned with the maximal correspondence of any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art. Amino acid sequence or nucleotide sequence that exhibits 80% homology, more preferably at least 90% homology, most preferably at least 95% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparisons using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to one embodiment of the invention, the polynucleotides encoding SDF-1α of the present invention comprises the sixth sequence.

According to one embodiment of the invention, the peripheral arterial disease of the present invention is ischemic limb disease (Ischemic Limb Disease). As demonstrated in the following examples, the composition of the present invention continued in steady state, unlike the leg condition of the pCK-administered group, the pCK-SDF-1a-administered group and the pCK-HGF-administered group worsened in a mouse model inducing lower limb ischemia. It is effective to keep it.

According to one embodiment of the present invention, the polynucleotide of the present invention is a nucleotide contained in naked DNA or a gene carrier. The compositions of the present invention can be applied in vivo through various delivery methods commonly known in the art of gene therapy, and such gene carriers include, but are not limited to, for example, vectors, plasmids and viral vectors.

(i) plasmid (vector)

Plasmids (vectors) can be used as carriers to carry the polynucleotides of the invention. The polynucleotides included in the vector are preferably present in a suitable expression cassette. The polynucleotide in the expression cassette is preferably operably linked to a promoter.

As used herein, the term “operably linked” refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences or transcriptional regulator binding sites) and other nucleic acid sequences, whereby the regulation The sequence will control the transcription and / or translation of said other nucleic acid sequence.

In the present invention, a promoter bound to a polynucleotide sequence can operate in an animal cell according to one embodiment, a mammalian cell according to another embodiment, and a human cell according to a specific embodiment, thereby controlling transcription of the nucleotide sequence, Promoters derived from mammalian viruses and promoters derived from genomes of mammalian cells, such as, for example, cytomegalo virus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 alpha promoter, metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene and human GM- Include, but are not limited to, promoters of the CSF gene is. According to one embodiment of the invention, the promoter used in the present invention is a promoter or EF1 alpha promoter derived from an IE (immediately early) gene of human CMV (hCMV), according to another embodiment a promoter / enhancer of CMV IE gene And 5′-UTR (untranslated region) comprising the entire exon 1 sequence up to just before the ATG start codon of exon 2.

Expression cassettes used in the present invention may comprise a polyaninated sequence, for example, from the ectopic growth hormone terminator (Gimmi, ER, et al., Nucleic Acids Res. 17: 6983-6998 (1989)), SV40 Poly adenylation sequence (Schek, N, et al., Mol. Cell Biol. 12: 5386-5393 (1992)), HIV-1 polyA (Klasens, BIF, et al., Nucleic Acids Res. 26: 1870-1876 (1998)), β-globin polyA (Gil, A., et al, Cell 49: 399-406 (1987)), HSV TK polyA (Cole, CN and TP Stacy, Mol. Cell. Biol. 5: 2104- 2113 (1985)) or polyomavirus polyA (Batt, D. B and GG Carmichael, Mol. Cell. Biol. 15: 4783-4790 (1995)).

According to another embodiment of the present invention, the gene carrier of the present invention may use a pCK, pCP, pVAX1 or pCY vector, and in accordance with certain embodiments of the present invention, a pCK vector may be used. The pCK vector is disclosed in detail in WO 2000/040737, the disclosure of which is incorporated herein by reference in its entirety.

(ii) retroviruses

Retroviruses are widely used as gene transfer vectors because they insert their genes into the host genome, carry large amounts of foreign genetic material, and have a broad spectrum of cells that can infect them.

To construct the retroviral vector, the polynucleotide sequence of the present invention is inserted into the retroviral genome instead of the sequence of the retrovirus to produce a nonreplicating virus. To produce virions, a packaging cell line comprising the gag, pol and env genes but no LTR (long terminal repeat) and ψ sequences is constructed (Mann et al., Cell, 33: 153-159 (1983)). When a recombinant plasmid comprising the polynucleotide sequence, LTR and ψ sequence of the present invention is introduced into the cell line, the ψ sequence enables the production of RNA transcripts of the recombinant plasmid, which transcript is packaged into a virus, and the virus is Discharged into the medium (Nicolas and Rubinstein "Retroviral vectors," In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513 (1988)). The medium containing the recombinant retrovirus is collected, concentrated and used as a gene delivery system.

Gene delivery using second generation retroviral vectors has been announced. Kasahara et al. (Science, 266: 1373-1376 (1994)) prepared a variant of the Moloney murine leuchemia virus, in which an EPO (erythropoietin) sequence was inserted into the envelope site to produce a chimeric protein with new binding properties. Produced. The polynucleotide sequences of the present invention can also be loaded into retroviruses according to this second generation retroviral vector construction strategy.

(iii) adenovirus

Adenoviruses are widely used as gene transfer vectors because of their medium genome size, ease of manipulation, high titers, wide range of target cells and excellent infectivity. Both ends of the genome contain 100-200 bp inverted terminal repeats (ITRs), which are cis-elements essential for DNA replication and packaging. The genome El region (E1A and E1B) encodes proteins that regulate transcription and transcription of host cell genes. The E2 regions (E2A and E2B) encode proteins that are involved in viral DNA replication.

Among the adenovirus vectors currently developed, non-replicating adenoviruses lacking the E1 region are widely used. On the other hand, the E3 region is removed from conventional adenovirus vectors to provide a site for insertion of foreign genes (Thimmappaya, B. et al., Cell, 31: 543-551 (1982); and Riordan, JR et al., Science, 245: 1066-1073 (1989)). Therefore, the polynucleotide sequence of the present invention is preferably inserted into the deleted E1 region (E1A region and / or E1B region) or E3 region. In addition, the nucleotide sequence can be inserted into the deleted E4 region. As used herein, the term "deletion" as used in connection with a viral genome sequence has the meaning including not only that sequence in its entirety, but also partially deleted. In addition, because adenovirus can pack up to about 105% of the wild-type genome, about 2 kb can be additionally packaged (Ghosh-Choudhury et al., EMBO J., 6: 1733-1739 (1987)). Thus, the above-described foreign sequence inserted into the adenovirus may additionally bind to the genome of the adenovirus.

Adenoviruses have 42 different serotypes and subgroups of A-F. Of these, adenovirus type 5 belonging to subgroup C is the most preferred starting material for obtaining the adenovirus vector of the present invention. Biochemical and genetic information for adenovirus type 5 is well known. Foreign genes carried by adenoviruses replicate in the same way as episomes, with very low genetic toxicity to host cells. Therefore, gene therapy using an adenovirus gene delivery system is considered to be safe.

(iv) AAV vector

Adeno-associated virus (AAV) is suitable as the gene delivery system of the present invention because it can infect non-dividing cells and has the ability to infect various kinds of cells. Details of the preparation and use of AAV vectors are disclosed in detail in US Pat. Nos. 5,139,941 and 4,797,368.

Studies on AAV as a gene delivery system are described in LaFace et al, Viology, 162: 483486 (1988), Zhou et al., Exp. Hematol. (NY), 21: 928-933 (1993), Walsh et al, J. Clin. Invest., 94: 1440-1448 (1994) and Flotte et al., Gene Therapy, 2: 29-37 (1995). Recently, AAV vectors have undergone clinical I as a therapeutic agent for cystic fibrosis.

Typically, AAV viruses are plasmids containing the gene sequence of interest, with two AAV terminal repeats flanked (McLaughlin et al., J. Virol., 62: 1963-1973 (1988); and Samulski et al. , J. Virol., 63: 3822-3828 (1989)) and expression plasmids comprising wild type AAV coding sequences without terminal repeats (McCarty et al., J. Virol., 65: 2936-2945 (1991)). Prepared by co-transfection.

(v) other viral vectors

Other viral vectors can also be used to carry the polynucleotide sequence of the present invention in vivo. Basinian virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999); Ridgeway, "Mammalian expression vectors," In: Vectors: A survey of molecular cloning vectors and their uses.Rodrigue and Denhardt, eds Stoneham: Butterworth, 467-492 (1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer.New York: Plenum Press, 117-148 (1986) and Coupar et al., Gene, 68: 1-10 (1988), lentiviruses (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 (1999)) or vectors derived from the herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92: 1411-1415 (1995)) can also carry the polynucleotide into cells. Available as a transport system.

(vi) liposomes

Liposomes are automatically formed by phospholipids dispersed in the aqueous phase. Examples of successfully delivering foreign DNA molecules into liposomes into cells include Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982) and Nicolau et al., Methods Enzymol., 149: 157-176 (1987). Liposomes containing the polynucleotide sequence of the present invention interact with cells through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with plasma cell membranes to transport the polynucleotide sequences into cells.

In the present invention, when the polynucleotide sequence of the present invention is mounted on naked recombinant DNA molecules or plasmids (vectors), the microinjection method (Capecchi, MR, Cell, 22: 479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094-1099 (1985)), calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456 (1973); and Chen and Okayama, Mol. Cell. Biol. 7: 2745 -2752 (1987)), electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982); and Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718 ( 1986)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982); and Nicolau et al , Methods Enzymol., 149: 157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)) and gene balm (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) can be used to import polynucleotide sequences into cells.

When the polynucleotide sequence of the present invention is constructed based on a viral vector, the polynucleotide sequence may be transported into cells according to various viral infection methods known in the art. Infection of host cells with viral vectors is described in the references cited above.

According to another embodiment of the invention, the gene carrier of the invention is a vector.

According to certain embodiments of the invention, the vector of the invention is a plasmid, and according to certain embodiments of the invention, the plasmid of the invention is pCK. Recombinant vectors comprising a single polynucleotide expressing two or more isoforms of HGF using pCK are described in detail in WO 2000/040737 and WO 2003/078568.

The composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. no. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

According to one embodiment of the invention, the pharmaceutical composition of the invention is administered parenterally. For example, intravenous administration, intraperitoneal administration, subcutaneous administration, intradermal administration, spinal cord administration, intrathecal administration, intraventricular administration, intraparenchymal administration, intracranial administration, intramuscular administration or topical administration may be used. According to another embodiment of the present invention, the pharmaceutical composition of the present invention can be administered intramuscularly, intrathecal, intrathecal, intraventricular, intraventricular, intracranial.

The pharmaceutical compositions of the invention can be formulated and administered by injection. Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, time of administration, route of administration, rate of excretion and response to reaction, Usually an experienced physician can easily determine and prescribe a dosage effective for the desired treatment.

According to one embodiment of the invention, the HGF, isoforms, and SDF-1α of the present invention are administered at a dosage of 10 ng to 100 mg, respectively, wherein the polynucleotides encoding the protein are each 1 μg to 100 mg It is administered at a dose of. The dosage may be the same or different each time when the administration of the HGF, isoforms thereof, SDF-1α or polynucleotides encoding it is repeated more than once.

The pharmaceutical compositions of the present invention are prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients, according to methods which may be readily practiced by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides (a) Hepatocyte Growth Factor (HGF) or a variant thereof, and Stromal Cell derived Factor 1 alpha (SDF-1α); Or (b) administering to a subject in need thereof a composition comprising a polynucleotide encoding HGF and a polynucleotide encoding SDF-1α as an active ingredient to a subject in need thereof. .

As used herein, the term “administering” or “administering” refers to the formation of the same amount in a subject's body by directly administering a therapeutically effective amount of a composition of the invention to an individual in need thereof (ie, a subject). Say what you can. Thus, the term "administration" includes the injection of a composition of the present invention into a lesion site, so the term "administer" is used in the same sense as "inject".

A “therapeutically effective amount” of a composition means a content of the composition that is sufficient to provide a therapeutic or prophylactic effect to the individual to whom the composition is to be administered, and includes a “prophylactically effective amount” as used herein. The term “individual” includes, without limitation, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey, specifically, an individual of the present invention is a human to be.

In the present invention, a method for preventing or treating peripheral arterial disease is a method comprising administering a pharmaceutical composition for preventing or treating peripheral arterial disease, which is an aspect of the present invention, and thus, overlapping descriptions thereof are excessive. It is omitted to avoid complexity.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for preventing or treating peripheral artery disease (PAD).

(b) Peripheral arterial disease using the composition of the present invention than the case of using polynucleotides encoding HGF, its heteromorphs, SDF-1α or its proteins through remarkable acceleration of vascular endothelial cell migration and blood vessel formation. For example, ischemic retardation disease) can be effectively prevented or treated.

1 shows the effect of the combination of HGF and SDF-1α on the cell lineage of HUVECs.

2A and 2B show the effect of the combination of HGF and SDF-1α on blood vessel production.

3 shows the effect of combination of pCK-HGF and pCK-SDF-1α on the leg status of the ischemic mouse model over time.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example  One. HUVEC (Human Umbilical vein endothelial cell) In Yuzhou  With HGF SDF - 1α  Experimental method to confirm efficacy of combination

HUVECs obtained by culturing single cells from cultured human umbilical cords and culturing single cells were purchased from Lonza.

HGF (SEQ ID NO: 1, R & D systems Cat No. 294-HG-025 / CF, USA) and SDF-1α (SEQ ID NO: 4, R & D systems Cat No. 350-NS-010 /) in the cell strain of HUVEC CF, USA) was coated with 1% gelatin transwell (Corning, cat # 3422) and then seeded 2 × 10 ⁴ cells per well. After culturing for one hour to allow the cells to adhere, the experimental groups were organized as follows (50 ng / ml HGF; 50 ng / ml SDF-1α; 25 ng / ml HGF + 25 ng / ml SDF-1α). Each experimental group was treated with the protein for two hours, and the cells were stained with crystal violet in order to measure the degree of cell migration, and then the number of cells moved through the transwell was measured using a microscope.

As a result, when treated with 50 ng / ml HGF alone, 1.8-fold cell line increased compared to the control, and treated with 50 ng / ml SDF-1α alone increased 1.5-fold cell line compared to the control. When HGF and SDF-1α were treated with 25 ng / ml each, the cell strain increased 2.5 times compared to the control group, and it was confirmed that the effect on the cell strain was better than that treated with each one alone (FIG. 1). .

Example  2. Matrigel  Plug Analysis ( Matrigel  in a plug assay With HGF SDF - 1α  Identify the efficacy of combinations on angiogenesis

Matrigel plug analysis was used to determine the effect of HGF and SDF-1α on blood vessel formation.

Five-week-old C57BL / 6 mice were divided into the following groups (PBS; 300 ng HGF; 150 ng HGF + 150 ng SDF-1α). One unit of heparin was added to a 400 μl Matrigel matrix (Corning, cat # 356231), and proteins corresponding to the experimental groups were added. The resulting Matrigel mixture was injected subcutaneously into the mouse abdomen. After 7 days, the mice were sacrificed and the transplanted Matrigel matrix was isolated, and the hemoglobin level contained in each Matrigel was measured by Drabkin's assay to quantify the degree of blood vessel formation.

As a result, the hemoglobin level of the group containing 300 ng HGF was about 1.8 times higher than that of the group containing PBS. The hemoglobin level of the group containing 150 ng of HGF and SDF-1α was higher than that of the PBS group. About 2.3-fold increase, it was confirmed that the degree of angiogenesis was improved compared to the administration of HGF alone (FIGS. 2A and 2B).

Example  3. lower limb ischemia ( hindlimb ischemia ; HLI ) Mouse model HGF  And SDF Confirmation of Dose Effect of -1α

Example  3-1: Preparation of plasmid DNA

pCK - HGF  Preparation of the plasmid

Prior to experiments with the ischemic mouse model of the lower limb, plasmid DNA to be used was prepared in the following manner. The pCK vector is a vector constructed so that the subject to be expressed is under the control of an enhancer / promoter of human cytomegalovirus (hCMV), and Lee et al., Biochem. Biophys. Res. Commun. 272: 230 (2000); It is disclosed in detail in WO 2000/040737. The pCK-HGF plasmid used in the present invention is a hybrid gene (i.e., HGF-X7) having a fragment sequence of intron 4 of the human HGF gene inserted between exons 4 and 5 of the human HGF gene according to the method described in WO 2003/078568. Gene; SEQ ID NO: 5) was inserted into the pCK vector.

pCK - SDF - 1α  Preparation of the plasmid

Genetic synthesis was performed by adding NheI and NotI restriction enzyme sequences at both ends based on human SDF-1α gene information (NCBI Reference Sequence: NM_199168.3). The synthesized human SDF-1α fragment was inserted into pCK vector using NheI and NotI. The sequence of the human SDF-1α gene inserted into the pCK vector is shown in SEQ ID NO: 6.

Example  3-2: lower limb ischemia ( HLI ) Mouse Model Preparation and Plasmid DNA Administration

The HLI mouse model is the most representative mouse model that mimics human critical Limb Ischemia (CLI) [1, 2]. The manufacturing method of the mouse model is as follows. Seven-week-old male Balb / c mice were anesthetized with a mixture of zoletil and rumpun, and the thigh-sided skin was incised approximately 1 cm. Then, the location of the femoral artery in the inner thigh (final artery) was found and tied the length of about 1 cm of the artery well with 6-0 thick thread, the tissue between them was cut out to remove the blood vessels. In this way, ischemic conditions can be induced by removing blood vessels that fall down the thighs. Simultaneously with HLI induction, the plasmid DNA to be evaluated was administered to muscles near the removed blood vessel. Thereafter, the incision was well closed and watched to see if the mouse recovered well from anesthesia.

The HLI mouse model was organized into 6 rats per group, each administered the following plasmids: 200 μg pCK; 200 μg pCK-HGF; 200 μg pCK-SDF-1α; 200 μg pCK-HGF + 200 μg pCK-SDF-1α) was administered. The condition of the legs was observed at 3, 10, 17, 22 and 27 days after dosing respectively and scored according to certain criteria and quantified. The criteria used were as follows [3]: 0 = steady state; 1 = necrosis of the toenail; 2 = necrosis of the toes; 3 = necrosis of the foot tissue.

As a result, in the pCK-administered group, the average condition of the legs gradually began to worsen after HLI induction, and the score after about two weeks rose to about 1.83. The pCK-HGF-treated group also scored 0.66-0.83 over time. On the other hand, in the pCK-HGF and pCK-SDF-1α combination group, the score remained 0 after induction of HLI (FIG. 3).

As a result, in the case of co-administration of HGF and SDF-1α, and co-administration of polynucleotides encoding HGF and SDF-1α through Examples 1-3, each protein or polynucleotide was administered alone. The endothelial cell vascular endothelial cell migration and angiogenesis-promoting effect and treatment effect for ischemic retardation disease were remarkable.

references

Limbourg, A., et al., Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat Protoc . 4 (12): p. 1737-46, 2009.

2. Couffinhal, T., et al., Mouse model of angiogenesis. Am J Pathol . 152 (6): p. 1667-79, 1998.

3. Clayton, JA, D. Chalothorn, and JE Faber, Vascular endothelial growth factor-A specifies formation of native collaterals and regulates collateral growth in ischemia. Circ Res . 103 (9): p. 1027-36, 2008.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (14)

1. (a) Hepatocyte Growth Factor (HGF) or a variant thereof, and Stromal Cell derived Factor 1α (SDF-1α); Or (b) a pharmaceutical composition for preventing or treating peripheral artery disease (PAD) comprising a polynucleotide encoding HGF and a polynucleotide encoding SDF-1α as an active ingredient.
2. The composition of claim 1, wherein the HGF comprises the amino acid sequence of SEQ ID NO: 1.
3. 10. The composition of claim 1, wherein the variant of HGF comprises full length HGF (flHGF) and deleted variant HGF (dHGF).
4. The composition of claim 3, wherein the full length HGF comprises the amino acid sequence of SEQ ID NO: 2, and the deleted variant HGF comprises the amino acid sequence of SEQ ID NO: 3.
5. The composition of claim 1, wherein the SDF-1α comprises an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 8.
6. According to claim 1, wherein the polynucleotide encoding the HGF is a sequence corresponding to exons 1 to 4 of the human HGF gene, intron 4 or a fragment sequence of the human HGF gene, and exons 5 to 18 of the human HGF gene A composition comprising a sequence.
7. The composition of claim 6, wherein the polynucleotide encoding the HGF is SEQ ID NO: 5.
8. The composition of claim 1, wherein the polynucleotide encoding SDF-1α comprises a nucleotide sequence of SEQ ID NO: 6.
9. The composition of claim 1, wherein the peripheral arterial disease is ischemic limb disease.
10. The composition of claim 1, wherein the polynucleotide is a nucleotide contained in a naked DNA or a gene carrier.
11. The composition of claim 10, wherein said gene carrier is a vector.
12. 12. The composition of claim 11, wherein said vector is a plasmid.
13. 13. The composition of claim 12, wherein the plasmid is pCK.
14. (a) Hepatocyte Growth Factor (HGF) or a variant thereof, and Stromal Cell derived Factor 1α (SDF-1α); Or (b) administering to a subject in need thereof a composition comprising a polynucleotide encoding HGF and a polynucleotide encoding SDF-1α as an active ingredient to a subject in need thereof.

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